To date there is no generally applicable, “ideal” way to prepare a cell or tissue sample, e.g. for immunohistochemistry or detection of a nucleic acid of interest, respectively. Fixation and the reversibility of negative effects introduced by the fixation processhave a major impact on the detectability of polypeptide antigens and nucleic acids, respectively, and on the reproducibility of the results obtained thereupon.
For successful immunostaining of an antigen in a cell or tissue sample at least three criteria have to be met: a) retention of the antigen at its original site, b) accessibility of the antigen and c) correct conformation/preservation of the antigen/epitope of interest. It appears that at present no fixation and/or detection procedure fully meets all these three criteria. For most procedures known in the art, best performance for one or two of these criteria often occurs at the expense of reduced performance in at least one other criterion.
Several fixatives are available and are routinely used by clinical pathology laboratories. These fixatives include glutardialdehyde, formaldehyde, and acetone, or other organic solvents. The vast majority of fixation procedures are based on the use of cross-linking agents, like formaldehyde. The fixative solution usually is an aqueous formaldehyde solution that includes sodium phosphates, formulated to provide buffering (minimal pH change following addition of a small amount of strong acid or base) to pH 7.2-7.6 and an approximately isotonic solution (a solution with the same or nearly the same osmotic pressure as that of mammalian extracellular fluids, isotonic solutions are based on physiological saline).
In order to perform many state-of-the-art procedures sample fixation has to be virtually perfect.
If fixation is incomplete, instead of fixation one merely observes the coagulation of proteins caused by the reagents and/or conditions used to dehydrate the sample. This may, for example, negatively impact the preservation of the sample tissues, or their morphology or impair their long term stability.
A prolonged formaldehyde fixation step may resort in the formation of cross-linked protein molecules that form a dense network. This network can impair the penetration of paraffin wax into the sample or/and the access of antibody molecules to epitomes of interest in the sample. As a result an antigen of interest may be reversibly or even irreversibly masked. Further an epitope may be chemically modified (“destroyed”) e.g., by reaction with formaldehyde.
In addition, it is known that the activity of most enzymes is impaired after formaldehyde fixation.
Fixation in formaldehyde is among the most widely used procedure in clinical pathology. The most likely primary reason that this reagent is used is that fixation with formaldehyde may trap an antigen of interest at the site that it occupies in the living organism. The methylene bridges introduced upon formaldehyde fixation also help to preserve the morphology of the cell or tissue in a given sample. These positive effects, however, occur at the expense of permeability, and fixation of the sample may, for example, cause: changes in the accessibility and/or conformation of an antigen/epitope of interest; damage to nucleic acids; and inactivation of enzymes of interest in the fixed sample.
Cross-linking due to formaldehyde fixation may mask or destroy epitopes, leading to a false negative immuno-staining result. This failure is more likely to occur when the primary immuno-reagent is a monoclonal antibody than when the primary immuno-reagent is a polyclonal antiserum. These problems with formaldehyde fixation have lead to many attempts to reduce or eliminate the negative effects of formaldehyde fixation; many of these attempts are chronicled in the scientific literature.
For long term storage a fixed cell or tissue sample usually has to be de-hydrated and embedded in an appropriate embedding medium. Paraffin embedding is usually preferable to either plastic embedding or cutting un-embedded specimens with a vibrating microtome or in a cryostat.
As illustrated above, all fixation procedures to a certain extent represent compromises of various kinds. Often optimal preservation of morphology goes to the expense of accessibility for an antibody or destruction of an antigen or of an epitope thereon.
However, and important to the present disclosure, not only is there a high variability introduced during preparation of a specimen, like its fixation or further processing like embedding with paraffin, probably even more variability is caused by the various modes and routes of regaining immunological reactivity or accessibility in detection of nucleic acids, i.e. in procedures known as antigen retrieval.
Despite the broad use and great utility of e.g. immunohistochemical methods or methods for detecting a nucleic acid of interest in a cell or tissue sample there is a great need for further improvements. Such improvements may, for example, relate to more gentle fixation of a cell or tissue sample, to improvements in antigen retrieval or/and to better comparability and reproducibility of results and maybe even include the possibility of using antibodies for which the corresponding antigen or epitope is destroyed in standard procedures, like fixation with formaldehyde.
Surprisingly, the inventors of the present disclosure have found that the use of bis-maleic anhydrides as a cross-linking agent in the preparation/fixation of a cell or tissue sample is of tremendous advantage and can and will lead to significant improvements regarding at least one or even several of the problems in the known art.